The analysis of complex mixtures of polynucleotides is important in many biological applications. In many situations, it is necessary to separate components of such mixtures to detect target polynucleotides of interest, to determine the relative amounts of different components, and to obtain nucleotide sequence information.
Electrophoresis provides convenient methods for analyzing polynucleotides. Typically, polynucleotides can be separated on the basis of length, due to differences in electrophoretic mobility. For example, in a matrix such as a crosslinked polyacrylamide, polynucleotides typically migrate at rates that are inversely proportional to polynucleotide length, due to size-dependent obstruction by the crosslinked matrix. In free solution, polynucleotides tend to migrate at substantially the same rates because of their substantially identical mass to charge ratios, so that it is difficult to distinguish different polynucleotides based on size alone. However, distinguishable electrophoretic mobilities can be obtained in free solution using polynucleotides that contain different charge/mass ratios, e.g., by attaching to the polynucleotides a polymer or other chemical entity having a charge/mass ratio that differs from that of the polynucleotides alone (See, e.g., U.S. Pat. No. 5,470,705).
When different polynucleotides can be separated based on length or molecular weight, detection can usually be accomplished using a single detectable label, such as a radioisotope, fluorophore, or other suitable conventional label. However, in complex mixtures or when different-sequence polynucleotides have similar or identical mobilities, it is preferable to use two or more detectable labels to distinguish different polynucleotides unambiguously.
In DNA sequencing, it is now conventional to use two or more (usually four) different fluorescent (or other suitable) labels to distinguish sequencing fragments that terminate with one of the four standard nucleotide bases (A, C, G and T, or analogs thereof). Such labels are usually introduced into the sequencing fragments using suitably labeled extension primers or by performing primer extension in the presence of nonextendable nucleotides that contain unique labels. Electrophoresis of the labeled products generates ladders of fragments that can be detected on the basis of elution time or band position.
Currently, in Sanger dideoxy sequencing using labeled terminators, an exonuclease minus DNA polymerase that has a mutation that decreases the discrimination against dideoxy-nucleotides is utilized. Such a mutation is utilized because non-mutated polymerases typically incorporate deoxynucleotides at a rate that is several hundred to several thousand times that of dideoxynucleotides, resulting in unacceptably low dideoxynucleotide incorporation or unacceptably high artifacts and background during detection. Examples of mutated polymerases conventionally utilized in Sanger dideoxy sequencing include, for example, Taq DNA polymerase (F667Y) and E. coli DNA polymerase (F762Y).
The problem of preferential incorporation of deoxynucleotides over dideoxynucleotides utilizing non-mutated polymerases in Sanger dideoxy sequencing, as well as other problems discussed herein, are obviated by the present teachings.